Studies carried out during the current grant period have presented compelling evidence that a number of inducible cytochrome P450s, including 1A1, 2B1, 2E1, 3A1/2, 2C6, 2C12, and 2D6 are targeted to both the mitochondrial and ER membrane compartments. Results also suggest that additional proteins of the oxidative and chemical stress pathways, such as GSTs and NOSi are also targeted to both the cytoplasmic and mitochondrial matrix compartments by the dual targeting mechanism. Two broad objectives of this competing renewal application are: 1) to further investigate the two alternate mechanisms of activating the cryptic mitochondrial targeting sequence and mechanisms of regulation of these pathways, and 2) to determine the physiological significance of P450 and non-P450 proteins targeted to mitochondria. The following specific aims are proposed to accomplish the goal: 1) Further characterize the role of cAMP mediated phosphorylation in mitochondrial targeting of P4502B1, 2E1 and 3A1 and also study the effects of protein modification on the catalytic function of P450s in the mitochondrial compartment. 2) Continue studies on the mode of interaction of Adx with the N-terminal truncated P4501Al and phosphorylated P4502B1, 3A1 and 2E1, and map the precise P450 domains involved in Adx binding. 3) investigate the mechanisms of targeting of non-P450 proteins, such as GSTalpha, GST A4-4, and NOSi using a combination of site specific mutagenesis, in vitro protein import in isolated mitochondria, and in vivo expression in COS cells. 4) Purification and characterization of endoprotease involved in the activation of the cryptic mitochondrial targeting signal of P4501A1, and also in the mitochondrial targeting of non P450 proteins, GSTA4-4 and NOSi. 5) Finally, the physiological significance of P450 and NOSi targeting to mitochondria will be investigated by overexpression of these proteins in COS and C6 glioma cells in countering the mitochondrial oxidative and chemical stress, and also effects on mitochondrial metabolic and biosynthetic functions.